Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations

Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pK_as. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK_as in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pK_a values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pK_as including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pK_as shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.     

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.     

Correlation between base-excision repair gene polymorphisms and levels of in-vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes

In vitro benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in cultured peripheral lymphocytes have been shown to be a phenotypic biomarker of individual’s DNA repair phenotype that is associated with cancer risk. In this study, we explored associations between genotypes of base-excision repair genes (PARP1 Val762Ala, APEX1 Asp148Glu, and XRCC1 Arg399Gln) and in vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes in 706 cancer-free non-Hispanic white subjects. We found that levels of BPDE-induced DNA adducts were significantly higher in ever smokers than in never smokers and that individuals with the Glu variant genotypes (i.e., Asp/Glu and Glu/Glu) exhibited lower levels of BPDE-induced DNA adducts than did individuals with the common Asp/Asp homozygous genotype (median RAL levels: 32.0 for Asp/Asp, 27.0 for Asp/Glu, and 17.0 for Glu/Glu, respectively; P _trend = 0.030). Further stratified analysis showed that compared with individuals with the common APEX1-148 homozygous Asp/Asp genotype, individuals with the APEX1-148Asp/Glu genotype or the Glu/Glu genotype had a lower risk of having higher-level adducts (adjusted OR = 0.60, 95% CI: 0.36–0.98 and adjusted OR = 0.47, 95% CI: 0.26–0.86, respectively; P _trend = 0.012) among smokers. Such an effect was not observed in non-smokers. However, there was no significant interaction between the APEX1 Asp148Glu polymorphism and smoking exposure in this study population (P = 0.512). Additional genotype-phenotype analysis found that the APEX1-148Glu allele had significantly increased expression of APEX1 mRNA in 270 Epstein-Barr virus-transformed lymphoblastoid cell lines, which is likely associated with more active repair activity. Our findings suggest that the functional APEX1-148Glu allele is associated with reduced risk of having high levels of BPDE-induced DNA adducts mediated with high levels of mRNA expression.     

Cloning,expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis

A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.     

Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site

The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structurally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attractive antifungal target. Here we identify a C-terminal catalytic domain of CaCet1p from residue 257 to 520 and characterize a manganese-dependent and cobalt-dependent NTPase activity intrinsic to CaCet1p. The NTPase can be exploited to screen in vitro for inhibitors. The amino acids that comprise the active site of CaCet1p were identified by alanine-scanning mutagenesis, which was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the phosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396, Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are located within the hydrophilic interior of an eight-strand β barrel of Cet1p. Each of the eight strands contributes at least one essential amino acid. The essential CaCet1p residues include all of the side chains that coordinate manganese and sulfate (i.e., γ phosphate) in the Cet1p product complex. These results suggest that the active site structure and catalytic mechanism are conserved among fungal RNA triphosphatases.     

Structural characterization of the heme‐based oxygen sensor,AfGcHK, its interactions with the cognate response regulator,and their combined mechanism of action in a bacterial two‐component signaling system

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109‐5 forms a two‐component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N‐terminal sensor domain causes the C‐terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen‐deuterium exchange coupled to mass spectrometry (HDX‐MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX‐MS studies on the AfGcHK:RR complex also showed that the N‐side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β‐strand B2 area of the RR protein's Rec1 domain, and that the C‐side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β‐strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375–1389. © 2016 Wiley Periodicals, Inc.     

Structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation

Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)₈ triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities.     

Crystal structure and biochemical studies of Brucella melitensis 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase

The prokaryotic 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6 Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron‐ and sulfur‐oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af‐Tth) was determined by X‐ray crystallography to a resolution of 1.95 Å. Af‐Tth is a homodimer, and its monomer structure exhibits an eight‐bladed β‐propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β‐propeller region. We observed unexplained electron densities in this cavity of the substrate‐soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site‐specific Af‐Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af‐Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.     

Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation.     

Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form,and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

The P9-1 protein of Rice black-streaked dwarf virus (RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed for in vitro binding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.     

Comparative In-Vitro Functional Analysis of Synthetic Defensins and Their Corresponding Peptide Variants Against HIV-1NL4.3, E. coli, S. aureus and P. aeruginosa

Defensins found in mammals belong to mainly two subfamilies α- and β-defensins. Mammalian defensins are small molecules (18–45 residues) that are cysteine, arginine rich compounds. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. To investigate the structure and activity relationship, amino acid substitutions that alter charge were introduced into synthetic defensin peptides by adding 2–2 Arg (RR) and Asp (DD) at both the terminal and tested their effects on HIV-1, E. coli, S. aureus, and P. aeruginosa. In the present study, we have chemically synthesized native defensin peptides and their variants with Arg (RR) and Asp (DD) amino acid residues at N- and C-termini. Later, we assayed their anti-HIV, anti-microbial activities, stability, cytotoxicity and hemolytic properties. We reported that anti-HIV and antimicrobial activities of native defensins is increased significantly by adding Arg (RR) residues at both the termini while the substitution of Arg (RR) with Asp (DD), eliminate anti-HIV and antimicrobial activity against all bacterial species tested. While other physical features i.e. stability, cell toxicity and hemolytic property were not affected by any of the changes in the sequence. The results suggest that the terminal residues in defensins are crucial functional elements that determine their microbicidal potency. The enhanced microbicidal activity observed for defensin peptides with Arg (RR) residues could be due to optimization of amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.     

Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe

RNA cap guanine-N2 methyltransferases such as Schizosaccharomyces pombe Tgs1 and Giardia lamblia Tgs2 catalyze methylation of the exocyclic N2 amine of 7-methylguanosine. Here we performed a mutational analysis of Giardia Tgs2, entailing an alanine scan of 17 residues within the minimal active domain. Alanine substitutions at Phe18, Thr40, Asp76, Asn103 and Asp140 reduced methyltransferase specific activity to <3% of wild-type Tgs2, thereby defining these residues as essential. Alanines at Pro142, Tyr148 and Pro185 reduced activity to 7–12% of wild-type. Structure–activity relationships at Phe18, Thr40, Asp76, Asn103, Asp140 and Tyr148, and at three other essential residues defined previously (Asp68, Glu91 and Trp143) were gleaned by testing the effects of 18 conservative substitutions. Our results engender a provisional map of the Tgs2 active site, which we discuss in light of crystal structures of related methyltransferases. A genetic analysis of S. pombe Tgs1 showed that it is nonessential. An S. pombe tgs1Δ strain grows normally, notwithstanding the absence of 2,2,7-trimethylguanosine caps on its U1, U2, U4 and U5 snRNAs. However, we find that S. pombe requires cap guanine-N7 methylation catalyzed by the enzyme Pcm1. Deletion of the pcm1⁺ gene was lethal, as were missense mutations in the Pcm1 active site. Thus, whereas m⁷G caps are essential in both S. pombe and S. cerevisiae, m^2,2,7G caps are not.     

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb–Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb–Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ ₁ dihedral combinations (g+, g+) and (g?, g+), respectively. These low energy conformations were also observed in intrahelical Asp–Arg and Glu–Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.     

Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.     

A Novel High-Molecular-Mass Bacteriocin Produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis>: Biochemical Features and Mode of Action

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.     

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting dsDNA phage, ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities do not significantly impact infection outcomes.     

Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic α/β/α sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218V AtRCCR, and substrate-free F218V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the β-sheet and the C-terminal α-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.